Dispersal of proteolipid macroaggregates with trifluoroacetic acid and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The propensity of highly purified proteolipids to form macroaggregates in aqueous solutions, especially when heated with sodium dodecyl sulfate (SDS), with or without thiol reagents, has made qualitative and quantitative analyses of individual species by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) difficult and unreliable. Comparisons of proteolipid profiles from liver, brain, and cultured human keratinocytes demonstrate that 40-72% of the total proteolipid in SDS-PAGE sample buffer is in the form of macroaggregates. Treatment of proteolipids with neat trifluoroacetic acid (TFA) followed by removal of the TFA and incubation in cold SDS-PAGE sample buffer causes complete dispersal of the macroaggregates and allows recovery of virtually all of the proteolipid applied to gels (increasing yields by as much as 3.6 times, depending on tissue type). Gels of TFA-treated samples display differences not only in the relative amounts of individual species but also in novel species not found in untreated samples. Eluted macroaggregates treated with TFA display the same SDS-PAGE banding profiles as TFA-treated whole proteolipids. Hence, routine TFA treatment of proteolipids prior to SDS-PAGE increases total proteolipid yields, allows reliable quantitation of individual apoprotein species, and reveals species previously obscured by the formation of macroaggregates.
Grayson
S
S
Department of Dermatology, University of California, San Francisco School of Medicine.
Sequeira
S M
SM
D000818:Animals; D003720:Densitometry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; D014269:Trifluoroacetic Acid / Q000737:chemistry*
D011510:Proteolipids; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; C048139:Coomassie blue; D014269:Trifluoroacetic Acid